Using single molecule magnetic tweezers to dissect titin energy release during muscle contraction

Mechanical forces regulate biological processes in unique and unexpected ways, but many biochemical methods are unable to reproduce the vectorial stretching experienced by proteins in cells. Force spectroscopy techniques remedy these shortcomings by utilizing microscopic force probes to stretch and relax single protein, DNA, and RNA molecules. The central focus of this thesis is the development and implementation of a custom-built protein magnetic tweezers for unfolding and refolding Ig domains from titin, a critical filament of the sarcomere, and the longest continuous peptide in the human body. Suspended from the Z-disc to the tip of the thick filament, titin sustains the brunt of intracellular forces during muscle elongation. Since the discovery of titin, it has been widely debated whether Ig domain unfolding contributes to muscle mechanics. A combination of single quantum dot tracking in myofibrils extracted from rabbit muscle and single molecule magnetic tweezers experiments on recombinant titin fragments confirms, for the first time, the presence of titin Ig domain unfolding and refolding at physiological sarcomere lengths and stretching forces. The magnetic tweezers experiments show the surprising ability of titin Ig domains to generate piconewton level forces during folding, and we advance the hypothesis that titin folding is an important source of energy during muscle contraction. Muscle elongation recruits Ig domains to the unfolded state, whereby folding is initiated through reduction of force on titin upon actomyosin crossbridges formation. Magnetic tweezers measurements demonstrate that titin Ig folding generates peak work, velocity, and power output of 64 zeptojoules, 1.9 microns per second, and 6,000 zeptowatts, matching or exceeding the equivalent single molecule measurements from single molecule myosin II powerstrokes. The forces generated by protein folding are therefore likely to be an integral part of the contractile process of animal muscle.